Lrp1 binding agents and uses thereof

ABSTRACT

Provided herein are agents that hind to binding domain I of LRP1 and mimic the activity of prosaposin in stimulating Tsp-1. Further provided herein are agents that inhibit the function (e.g., the ability to repress Tsp-1) of Protease, Serine 2 (PRSS2) by inhibiting the binding of PRSS2 to LRP1. Methods of using these agents in treating cancer are also provided.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/475,133 filed Mar. 22, 2017 and entitled“LRP1 BINDING AGENTS AND USES THEREOF”, the entire contents of which areincorporated by reference herein.

BACKGROUND

The current standard of care for cancer patients consists of broadlyacting cytotoxic agents (chemotherapy), radiation, and directedtherapeutics that target specific secreted proteins, cell surfacereceptors, or kinases. Historically, there have been two classes oftherapeutics that target the tumor microenvironment, anti-angiogenicdrugs (which have been limited to anti-VEGF therapies) andimmunomodulatory drugs. One of the major drawbacks to therapies thattarget the microenvironment is that they do not have direct anti-tumoractivity and thus their efficacy as monotherapies has been limited.Conversely, a major drawback of targeted therapies and chemotherapiesthat have direct anti-tumor activity is that patients develop resistanceto the drug, in addition to unintended deleterious side effects.

SUMMARY

Provided herein are novel cancer therapeutic strategies that possessboth anti-cancer activity and target the cancer microenvironment toprevent cancer reoccurrence and/or metastasis, by stimulating theactivity of the potent anti-angiogenic and anti-tumorigenic proteinThrombospondin 1 (Tsp-1).

Some aspects of the present disclosure provide methods of treatingcancer, the method comprising administering to a subject in need thereofan effective amount of an agent that binds to binding domain I of LowDensity Lipoprotein Receptor-related Protein 1 (LRP1).

In some embodiments, binding domain I of LRP1 comprises amino acids1-172 of LRP1. In some embodiments, the agent is a protein or peptide.

In some embodiments, the agent is an antibody. In some embodiments, theantibody is a polyclonal antibody. In some embodiments, the antibody isa monoclonal antibody. In some embodiments, the antibody hinds to aminoacids 151-172 (SEQ ID NO: 3) in the binding domain I of LRP1.

In some embodiments, the agent is a small molecule. In some embodiments,the small molecule is selected from the group consisting of: lipids,monosaccharides, second messengers, metabolites, and xenobiotics.

In some embodiments, binding of the agent to binding domain I of LRP1activates a Rho-GTPase pathway. In some embodiments, the Rho pathway isa LRP-1 mediated Rho-GTPase pathway. In some embodiments, binding of theagent to binding domain I of LRP1 stimulates Thrombospondin 1 (Tsp-1).

In some embodiments, the agent is administered orally, parenterally,intramuscularly, intranasally, intratracheally,intracerebroventricularly, intravenously, or intraperitoneally.

In some embodiments, the cancer is metastatic. In some embodiments, thecancer is biliary tract cancer; bladder cancer; brain cancer;glioblastoma; medulloblastoma; breast cancer; cervical cancer;choriocarcinoma; colon cancer; endometrial cancer: esophageal cancer;gastric cancer; hematological neoplasm; acute lymphocytic andmyelogenous leukemia; multiple myeloma; AIDS-associated leukemias andadult T-cell leukemia, lymphoma; intraepithelial neoplasm; Bowen'sdisease; Paget's disease; liver cancer; lung cancer; lymphomas;Hodgkin's disease; lymphocytic lymphoma; neuroblastomas; oral cancer;squamous cell carcinoma; ovarian cancer; pancreatic cancer; prostatecancer; rectal cancer; sarcomas; leiomyosarcoma; rhabdomyosarcoma;liposarcoma; fibrosarcoma; osteosarcoma; skin cancer; testicular cancer;stromal tumors and germ cell tumors; thyroid cancer; and renal cancer.In some embodiments, the cancer is prostate cancer, breast cancer,ovarian cancer, or pancreatic cancer.

Other aspects of the present disclosure provide antibodies bind bindingdomain I of Low Density Lipoprotein Receptor-related Protein 1 (LRP1).In some embodiments, binding domain I of LRP-1 comprises the amino acids1-172 of LRP-1. In some embodiments, the antibody binds to amino acids151-172(SEQ ID NO: 3) in binding domain I of LRP1.

In some embodiments, the antibody is a polyclonal antibody. In someembodiments, the antibody is a monoclonal antibody. In some embodiments,the antibody stimulates Tsp-1.

Further provided herein are methods of treating cancer, the methodcomprising administering to a subject in need thereof an effectiveamount of an agent that stimulates Thrombospondin 1 (Tsp-1). In someembodiments, the agent inhibits the ability of Protease, serine 2(PRSS2) to repress Tsp-1. In some embodiments, the agent binds tobinding domain I of LRP1. In some embodiments, the agent inhibitsbinding of PRSS2 to Low density lipoprotein receptor-related protein 1(LRP1).

Further provided herein are methods of treating cancer, the methodcomprising administering to a subject in need thereof an effectiveamount of a first agent that inhibits the function of Protease, serine 2(PRSS2), and an effective amount of a second agent that binds to bindingdomain I of Low density lipoprotein receptor-related protein I (LRP1).In some embodiments, the first agent and the second agent areadministered simultaneously. In some embodiments, the first agent andthe second agent are administered sequentially.

The details of one or more embodiments of the disclosure are set forthin the description below. Other features or advantages of the presentdisclosure will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein. In the drawings:

FIG. 1. Plot of average tumor mass of AsPc1 tumors treated with vehiclecontrol or peptide at a dose of 20 and 40 mg/kg/day.

FIGS. 2A-2B. Representative images of (FIG. 2A) primary pancreatictumors and (FIG. 2B) the lung and spleen of mice treated with psaptideor vehicle control.

FIGS. 3A-3B. Western blot of CD36 and β-actin in (FIG. 3A) 9 ovariancancer cell lines established from patient ascites and (FIG. 3B) breastcancer cell lines MCF7, SkBr3 and MDA-MB-231.

FIG. 4. SCID mice were injected with 1×10 ovarian cancer cells andtreated with peptide (40 mg/kg/QD) or cisplatin (4 mg/kg QOD)(n=12/group),

FIG. 5. FACS analysis of Gr1+ (x-axis) and CD11b+ (y-axis) cells in theascites of mice that were treated with vehicle control or the peptide(psaptide).

FIGS. 6A-6B. Western blot analysis of: (FIG. 6A) Tsp-1 and β-actinexpression in prostate fibroblasts that were untreated (−) or treatedwith conditioned media from PC3 cells alone or in combination with RAP,and (FIG. 6B) Tsp-1, p53 and β-actin expression in prostate fibroblaststhat were untreated (−) or treated with conditioned media from PC3 cellsalone or in combination with the PKC inhibitor Gδ 6983 (PKCi).

FIG. 7. Western blot analysis of Tsp-1, p53 and β-actin expression inMRC5 lung fibroblasts that were transduced with an empty vector (pLKO)or lentiviral vectors expressing two independent shRNA sequencesspecific for LRP1, which were untreated (−) or treated with conditionedmedia from PC3 cells or PC3M-LN4 cells.

FIGS. 8A-8B. Western blot analysis of Tsp-1 and β-actin expression inlung fibroblasts, which were (FIG. 8A) untreated (−) or treated withpsap peptide alone or in combination with 25 or 50 μg of rhRAP, and(FIG. 8B) untreated (−) or treated with psap peptide alone or withY27632.

FIG. 9. Western blot analysis of Tsp-1 and β-actin expression in lungfibroblasts that were untreated (−) or treated with LN4 CM fractionatedover a Cu^(2|)/heparin sepharose column with increasing concentrationsof NaCl.

FIG. 10. Western blot analysis of PRSS2 and β-actin expression in PC3and PC3M-LN4 cells.

FIG. 11. Western blot analysis of Tsp-1 and β-actin expression in lungfibroblasts that were untreated (−) or treated with LN4 CM alone or incombination with STI.

FIG. 12. Western blot analysis of Tsp-1 and β-actin expression in lungfibroblasts that is were untreated (−) or treated with LN4 CM alone orin combination with Rac1 inhibitor.

FIGS. 13A-13B. (FIG. 13A) Schematic diagram of miniLRP1 receptors. (FIG.13B) Western blot analysis of Tsp-1, miniLRP and β-actin expression in293T cells that were untreated (−) or treated the cyclic prosaposinpeptide.

FIGS. 14A-14B. Prosaposin and PRSS2 both require LRP1 for modulation ofTsp-1 expression.

FIGS. 15A-15C. Silencing PRSS2 blocks Tsp-1 repression and tumorformation.

FIGS. 16A-16B. Co-immunoprecipitation of LRP1 peptides with PSAP (FIG.16A) or PRSS2 (FIG. 16B).

FIG. 17. Mutations in the active site of PRSS2 do not affect therepression of Tsp-1. Mutations were made in the PRSS2 active site thatwould abolish the enzymatic activity of PRSS2. The mutants weresequenced to confirm the presence of the mutation. The wild-type andmutant PRSS2 proteins were ectopically transfected in 293T cells and theconditioned media was used to treat WI-38 fibroblasts. Tsp-1 and β-actinexpression was then analyzed by western blot. It was found that theability of PRSS2 to repress Tsp-1 was not affected, indicating thatbinding site for LRP1 is not in the active site and that antibodiesagainst this region do not affect the protease activity of the enzyme.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Provided herein are novel cancer therapeutic strategies that possessboth anti-cancer activity and ability to target the cancermicroenvironment to prevent cancer reoccurrence and/or metastasis. Theanti-cancer strategies described herein rely, at least in part, onstimulating the activity of a potent anti-angiogenic andanti-tumorigenic protein, Thrombospondin 1 (Tsp-1). “Tsp-1” is a subunitof a disulfide-linked homotrimeric protein. Tsp-1 is an adhesiveglycoprotein that mediates cell-to-cell and cell-to-matrix interactions.Tsp-1 binds to fibrinogen, fibronectin, laminin, type V collagen andintegrins alpha-V/beta-1 and has been shown to play roles in plateletaggregation, angiogenesis, and tumorigenesis. For the purpose of thepresent disclosure. Tsp-1 is a potent anti-tumorigenic andanti-angiogenic factor, whose activation suppresses tumor growth andmetastasis and represses angiogenesis in the tumor microenvironment.

Prosaposin or prosaposin-derived peptides were previously shown to beable to stimulate the activity of Tsp-1 and are effective for treatingmultiple types of cancers (see, e.g., PCT publications WO2009002931WO/2011/084685 and WO12013/096868, WO2015148801 and U.S. patentapplication Ser. Nos. 12/640,788 and 13/516,511, all of which areincorporated herein by reference in their entirety).

The present disclosure is based, at least in part, on the finding thatthe stimulating activity of prosaposin or a prosaposin-derived peptideto Tsp-1 is mediated by the Low Density Lipoprotein Receptor RelatedProtein 1 (LRP1.). “Low Density Lipoprotein Receptor Related Protein 1(LRP1),” also known as alpha-2-macroglobulin receptor (A2MR),apolipoprotein E receptor (APOER) or cluster of differentiation 91(CD91), is a protein forming a receptor found in the plasma membrane ofcells involved in receptor-mediated endocytosis. In humans, LRP1 isencoded by the LRP1 gene. LRP1 is a key signaling protein and isinvolved in various biological processes, such as lipoprotein metabolismand cell motility, and diseases, such as neurodegenerative diseases,atherosclerosis, and cancer.

The LRP1 gene encodes a 600 kDa precursor protein that is processed byfurin in the trans-Golgi complex, resulting in a 515 kDa alpha-chain andan 85 kDa beta-chain associated noncovalently. As a member of the LDLRfamily, LRP1 contains cysteine-rich complement-type repeats, EGF (gene)repeats, β-propeller domains, a transmembrane domain, and a cytoplasmicdomain. The cytoplasmic domain of LRP1 is the alpha-chain, whichcomprises four ligand-binding domains (termed “binding domains I-IV”)containing two, eight, ten, and eleven cysteine-rich complement-typerepeats, respectively. These repeats bind extracellular matrix proteins,growth factors, proteases, protease inhibitor complexes, and otherproteins involved in lipoprotein metabolism. Of the four domains, II andIV bind the majority of the protein's ligands. The EGF repeats andβ-propeller domains serve to release ligands in low pH conditions, suchas inside endosomes, with the β-propeller postulated to displace theligand at the ligand binding repeats. The transmembrane domain is theβ-chain, which contains a 100-residue cytoplasmic tail. This tailcontains two NPxY motifs that are responsible for the protein's functionin endocytosis and signal transduction.

To date, no ligand has been identified that binds to binding domain I ofLRP 1, described herein, binding domain I of LRP1 comprises about aminoacids 1-200 of LRP1 and contains the first two β-propeller domains ofLRP1. “About” means the binding domain I of LRP1 may be no more than 15%longer or shorter than 200 amino acids. For example, the binding domainI of LRP1 may comprise amino acids 1-200, 1-199, 1-198, 1-197, 1-196,1-195, 1-194, 1-193, 1-192, 1-191, 1-190, 1-189, 1-188, 1-187, 1-186,1-185, 1-184, 1-183, 1-182, 1-181, 1-180, 1-179, 1-178, 1-177, 1-176,1-175, 1-174, 1-173, 1-172, 1-171, 1-170, 1-201, 1-202, 1-203, 1-204,1-205, 1-206, 1-207, 1-208, 1-209, 1-210, 1-211, 1-212, 1-213, 1-214,1-215, 1-216, 1-217, 1-218, 1-219, 1-220, 1-221, 1-222, 1-223, 1-224,1-225, 1-226, 1-227, 1-228, 1-229, or 1-230 of the LRP1 protein. In someembodiments, the binding domain I of LRP1 comprises amino acids 1-172 ofthe LRP1 protein.

The present disclosure further provides the identification of ligandsthat bind to binding domain I of LRP1. As demonstrated in the Figuresand Examples of the present disclosure, prosaposin or prosaposin-derivedpeptides were found to bind to the binding domain I of LRP1 and thebinding of the prosaposin-derived peptide activates Tsp1 and repressescancer (e.g., FIGS. 13A and 13B). Another ligand of the binding domain Iof LRP1 identified in the present disclosure is Protease, serine 2(PRSS2) (e.g., FIG. 10). “PRSS2” is a trypsinogen and is a member of thetrypsin family of serine proteases. PRSS2 is secreted by the pancreasand cleaved to its active form in the small intestine. It is active onpeptide linkages involving the carboxyl group of lysine or arginine. Asdemonstrated herein, PRSS2 represses the activity of Tsp-1 in the tumormicroenvironment and thus promotes the progression of cancer. Theability of PRSS2 to repress Tsp-1 is mediated by binding of PRSS2 tobinding domain I of the LRP-1 protein.

Accordingly, some aspects of the present disclosure provide agents thatbind to the binding domain I of LRP1. Such agents mimic the activity ofprosaposin or prosaposin-derived peptides and activates Tsp-1 when boundto binding domain I of LRP1. in some embodiments, the agent binds to apeptide within amino acids 1-172 of the LRP1 protein. In someembodiments, the agent binds to a peptide within the region of aminoacids 140-180 of the LRP1 protein. For example, without limitation, theagent may bind to amino acids 140-180, 140-179, 140-178, 140-177,140-176, 140-175, 140-174, 140-173, 140-172, 140-171, 140-170, 140-169,140-168, 140-167, 140-166, 140-165, 140-164, 140-163, 140-162, 140-161,140-160, 141-180, 141-179, 141-178, 141-177, 141-176, 141-175, 141-174,141-173, 141-172, 141-171, 141-170, 141-169, 141-168, 141-167, 141-166,141-165, 141-164, 141-163, 141-162, 141-161, 142-180, 142-179, 142-178,142-177, 142-176, 142-175, 142-174, 142-173, 142-172, 142-171, 142-170,142-169, 142-168, 142-167, 142-166, 142-165, 142-164, 142-163, 142-162,143-180, 143-179, 143-178, 143-177, 143-176, 143-175, 143-174, 143-173,143-172, 143-171, 143-170, 143-169, 143-168, 143-167, 143-166, 143-165,143-164, 143-163, 144-180, 144-179, 144-178, 144-177, 144-176, 144-175,144-174, 144-173, 144-172, 144-171, 144-170, 144-169, 144-169, 144-168,144-167, 144-165, 144-164, 145-180, 145-179, 145-178, 145-177, 145-176,145-175, 145-174, 145-173, 145-172, 145-171, 145-170, 145-169, 145-168,145-167, 145-166. 145-165, 146-180, 146-179, 146-178, 146-177, 146-176,146-175, 146-174, 146-173, 146-172, 146-171, 146-170, 146-169, 146-168,146-167, 146-166, 147-180, 147-179, 147-178, 147-177, 147-176, 147-175,147-174, 147-173, 147-172, 147-171, 147-170, 147-169, 147-168, 147-167,148-180, 148-179, 148-178, 148-177, 148-176, 148-175, 148-174, 148-173,148-172, 148-171, 148-170, 148-169, 148-168, 149-180, 149-179, 149-178,149-177, 149-176, 149-175, 149-174, 149-173, 149-172, 149-171, 149-170,149-169, 150-180, 150-179, 150-178, 150-177, 150-176, 150-175, 150-174,150-173, 150-172, 150-171, 150-170, 150-170, 151-180, 151-179, 151-178,151-177, 151-176, 151-175, 151-174, 151-173, 151-172, 151-171, 152-180,152-179, 152-178, 152-177, 152-176, 152-175, 152-174, 152-173, 152-172,153-180, 153-179, 153-178, 153-177, 153-176, 153-175, 153-174, 153-173,153-172, 154-180, 154-179, 154-178, 154-177, 154-176, 154-175, 154-174,154-173, 154-172, 156-180, 156-179, 156-178, 156-177, 156-176, 156-175,156-174, 156-173, 156-172, 156-180, 156-179, 156-178, 156-177, 156-176,156-175, 156-174, 156-173,156-172, 157-180, 157-179, 157-178, 157-177,157-176, 157-175, 157-174, 157-173, 157-172, 158-180, 158-179, 158-178,158-177, 158-176, 158-175, 158-174, 158-173, 158-172, 159-180, 159-179,159-178, 159-177, 159-176, 159-175, 159-174, 159-173, 159-172, 160-180,160-179, 160-178, 160-177, 160-176, 160-175, 160-174, 160-173, or160-172 of the LRP 1 protein.

In some embodiments, the agent binds to amino acids 151-172 of the LRP1protein. For example, the agent may bind to amino acids 151-172,151-171, 151-170,151-169, 151-168, 151-167, 151-166, 151-165, 151-164,151-163, 151-162, 151-161, 151-160, 152-172, 152-171, 152-170,152-169,152-168, 152-167, 152-166, 152-165, 152-164, 152-163, 152-162, 152-161,153-172, 153-171, 153-170,153-169, 153-168, 153-167, 153-166, 153-165,153-164, 153-163, 153-162, 154-172, 154-171, 154-170,154-169, 154-168,154-167, 154-166, 154-165, 154-164, 154-163, 155-172, 155-171,155-170,155-169, 155-168, 155-167, 155-166, 155-165, 155-164, 156-172,156-171, 156-170,156-169, 156-168, 156-167, 156-166, 156-165, 156-164,157-172, 157-171, 157-170,157-169, 157-168, 157-167, 157-166, 158-172,158-171, 158-170,158-169, 158-168, 158-167, 159-172, 159-171,159-170,159-169, 159-168, 160-172, 160-171, 160-170, 160-169, 161-172,161-171, 161-170, 162-172, 162-171, or 163-172 of the LRP1 protein.

In some embodiments, the agent binds to amino acids 140-164 in thebinding domain of the LRP1 protein. For example, the agent may bind toamino acids 140-164, 140-163, 140-162, 140-161, 140-160, 140-159,140-158, 140-157, 140-156, 140-155, 140-154, 140-153, 140-152, 140-151,140-150, 141-164, 141-163, 141-162, 141-161, 141-160, 141-159, 141-158,141-157, 141-156,141-155, 141-154,141-153, 141-152, 141-151, 142-164,142-163, 142-162, 142-161, 142-160, 142-159, 142-158, 142-157, 142-156,142-155, 142-154, 142-153, 142-152, 143-164, 143-163, 143-162, 143-161,143-160, 143-159, 143-158, 143-157, 143-156, 143-155, 143-154, 143-153,144-164, 144-163, 144-162, 144-161, 144-160, 144-159, 144-158, 144-157,144-156, 144-155, 144-154, 145-164, 145-163, 145-162, 145-161, 145-160,145-159, 145-158, 145-157, 145-156, 145-155, 146-164, 146-163, 146-162,146-161, 146-160, 146-159, 146-158, 146-157, 146-156, 147-164, 147-163,147-162, 147-161, 147-160, 147-159, 147-158, 147-157, 148-164, 148-163,148-162, 148-161, 148-160, 148-159, 148-158, 149-164, 149-163, 149-162,149-161, 149-160, 149-159, 150-164, 150-163, 150-162, 150-161, 150-160,151-164, 151-163, 151-162, 151-161, 152-164, 152-163, 152-162, 153-164,153-163, or 154-164 of the LRP1 protein.

In some embodiments, the agent binds to amino acids 151-164 in thebinding domain of the LRP1 protein. For example, the agent may bind toamino acids 151-164, 151-163, 151-162, 151-161, 151-160, 151-159,151-158, 151-157, 151-156, 152-164, 152-163, 152-162, 152-161, 152-160,152-159, 152-158, 152-157, 153-164, 153-163, 153-162,153-161, 153-160,153-159, 153-158, 154-164, 154-163, 154-162, 154-161, 154-160, 154-159,155-164, 155-163, 155-162, 155-161, 155-160, 156-164, 156-163, 156-162,156-161, 157-164, 157-163, 157-162, 158-164, 158-163, or 159-164 of theLRP1 protein.

In some embodiments, the agent may be a mixture of agents that binddifferent portions of the binding domain I (e.g., amino acids 1-172) ofthe LRP1 protein.

The amino acid sequences of full-length LRP1, amino acids 1-172 of LRP1,amino acids 140-164 of LRP-1, amino acids 151-172 of LRP1, and aminoacids 151-164 of LRP1 are provided. The amino acid numbers providedherein are corresponding to the full-length LRP1 protein. One skilled inthe art is able to ascertain the position and the amino acid sequence ofthe peptide that is bound by the agent described herein.

Full length LRP1 (SEQ ID NO: 1, sequence does notcontain the N-terminal 20 amino acid signalsequence, which is removed after translation of the protein)IDAPKTCSPKQFACRDQITCISKGWRCDGERDCPDGSDEAPEICPQSKAQRCQPNEHNCLGTELCVPMSRLCNGVQDCMDGSDEGPHCRELQGNCSRLGCQHHCVPTLDGPTCYCNSSFQLQADGKTCKDFDECSVYGTCSQLCTNTDGSFICGCVEGYLLQPDNRSCKAKNEPVDRPPVLLIANSQNILATYLSGAQVSTITPTSTRQTTAMDFSYANETCWVHVGDSAAQTQLKCARMPGLKGFVDEHTINISLSLHHVEQMAIDWLTGNFYFVDDIDDRIFVCNRNGDTCVTLLDLELYNPKGIALDPAMGKVFFTDYGQIPKVERCDMDGQNRTKLVDSKIVFPHGITLDLVSRLVYWADAYLDYIEVVDYEGKGRQTIIQGILIEHLYGLTVFENYLYATNSDNANAQQKTSVIRVNRFNSTEYQVVTRVDKGGALHIYHQRRQPRVRSHACENDQYGKPGGCSDICLLANSHKARTCRCRSFGSLGSDGKSCKKPEHELFLVYGKGRPGIIRGMDMGAKVPDEHMIPIENLMNPRALDFHAETGFIYFADTTSYLIGRQKIDGTERETILKDGIHNVEGVAVDWMGDNLYWTDDGPKKTISVARLEKAAQTRKTLIEGKMTHPRAIVVDPLNGWMYWTDWEEDPKDSRRGRLERAWMDGSHRDIFVTSKTVLWPNGLSLDIPAGRLYWVDAFYDRIETILLNGTDRKIVYEGPELNHAFGLCHHGNYLFWTEYRSGSVYRLERGVGGAPPTVTLLRSERPPIFEIRMYDAQQQQVGTNKCRVNNGGCSSLCLATPGSRQCACAEDQVLDADGVTCLANPSYVPPPQCQPGEFACANSRCIQERWKCDGDNDCLDNSDEAPALCHQHTCPSDRFKCENNRCIPNRWLCDGDNDCGNSEDESNATCSARTCPPNQFSCASGRCIPISWTCDLDDDCGDRSDESASCAYPTCFPLTQFTCNNGRCININWRCDNDNDCGDNSDEAGCSHSCSSTQFKCNSGRCIPEHWTCDGDNDCGDYSDETHANCTNQATRPPGGCHTDEFQCRLDGLCIPLRWRCDGDTDCMDSSDEKSCEGVTHVCDPSVKGFCKDSARCISKAWVCDGDNDCEDNSDEENCESLACRPPSHPCANNTSVCLPPDKLCDGNDDCGDGSDEGELCGQCSLNNGGCSHNCSVAPGEGIVCSCPLGMELGPDNHTCQIQSYCAKHLKCSQKCDQNKFSVKCSCYEGWVLEPDGESCRSLDPFKPFIIFSNRHEIRRIDLHKGDYSVLVPGLRNTIALDFHLSQSALYWTDVVEDKIYRGKLLDNGALTSFEVVIQYGLATPEGLAVDWIAGNIYWVESNLDQIEVAKLDGTLRTTLLAGDIEHPRAIALDPRDGILFWTDWDASLRPRIEAASMSGAGRRTVHRETGSGGWPNGLTVDYLEKRILWIDARSDAIYSARYDGSGHMEVLRGHEFLSHPFAVTLYGGEVYWTDWRTNTLAKANKWTGHNVTVVQRTNTQPFDLQVYHPSRQPMAPNPCEANGGQGPCSHLCLINYNRTVSCACPHLMKLHKDNTTCYEFKKFLLYARQMEIRGVDLDAPYYNYIISFTVPDIDNVTVLDYDAREQRVYWSDVRTQAIKRAFINGTGVETVVSADLPNAHGLAVDWVSRNLFWTSYDTNKKQINVARLDGSFKNAVVQGLEQPHGLVVHPLRGKLYWTDGDNISMANMDGSNRTLLFSGQKGPVGLAIDFPESKLYWISSGNHTINRCNLDGSGLEVIDAMRSQLGKATLALAIMGDKLWWADQVSEKMGTCSKADGSGSVVLRNSTTLVMHMKVYDESIQLDHKGTNPCSVNNGDCSQLCLPTSETTRSCMCTAGYSLRSGQQACEGVGSFLLYSVHEGIRGIPLDPNDKSDALVPVSGTLSAVGIDFHAENDTIYWVDMGLSTISRAKRDQTWERDVVTNGIGRVEGAIVDWIAGNIYWTDQGFDVEIVARLNGSFRYVVISQGLDKPRAITVHPEKGYLFWTEWGQYPRIERSRLDGTERVVLVNVSISWPNGISVDYQDGKLYWCDARTDKIERIDLETGENREVVLSSNNMDMFSVSVFEDFIYWSDRTHANGSIKRGSKDNATDSVPLRTGIGVQLKDIKVFNRDRQKGTNVCAVANGGCQQLCLYRGRGQRACACAHGMLAEDGASCREYAGYLLYSERTILKSIHLSDERNLNAPVQPFEDPEHMKNVAILAFDYRAGTSPGTPNRIFFSDIHFGNIQQINDDGSRRITIVENVGSVEGLAYHRGWDTLYWTSYTTSTITRHTVDQTRPGAFERETVITMSGDDHPRAFVLDECQNLMFWTNWNEQHPSIMRAALSGANVLTLIEKDIRTPNGLAIDHRAEKLYFSDATLDKIERCEYDGSHRYVILKSEPVHPFGLAVYGEHIFWTDWVRRAVQRANKHVGSNMKLLRVDIPQQPMGIIAVANDTNSCELSPCRINNGGCQDLCLLTHQGHVNCSCRGGRILQDDLTCRAVNSSRAQDEFECANGECINFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQCSNGRCVSNMLWCNGADDCGDGSDEIPCNKTACGVGEFRCRDGTCIGNSRRCNQFVDCEDASDEMNCSATDCSSYFRLGVKGVLFQPCERTSLCYAPSWVCDGANDCGDYSDERDCPGVKRPRCPLNYFACPSGRCIPMSWTCDKEDDCEHGEDETHCNKFCSEAQFECQNHRCISKQWLLCDGSDDCGDGSDEAAHCEGKTCGPSSFSCPGTHVCVPERWLCDGDKDCADGADESIAAGCLYNSTCDDREFMCQNRQCIPKHFVCDHDRDCADGSDESPECEYPTCGPSEFRCANGRCLSSRQWECDGENDCHDQSDEAPKNPHCTSQEHKCNASSQFLCSSGRCVAEALLCNGQDDCGDSSDERGCHINECLSRKLSGCSQDCEDLKIGFKCRCRPGFRLKDDGRTCADVDECSTTFPCSQRCINTHGSYKCLCVEGYAPRGGDPHSCKAVTDEEPFLIFANRYYLRKLNLDGSNYTLLKQGLNNAVALDFDYREQMIYWTDVTTQGSMIRRMHLNGSNVQVLHRTGLSNPDGLAVDWVGGNLYWCDKGRDTIEVSKLNGAYRTVLVSSGLREPRALVVDVQNGYLYWTDWGDHSLIGRIGMDGSSRSVIVDTKITWPNGLTLDYVTERIYWADAREDYIEFASLDGSNRHVVLSQDIPHIFALTLFEDYVYWTDWETKSINRAHKTTGTNKTLLISTLHRPMDLHVFHALRQPDVPNHPCKVNNGGCSNLCLLSPGGGHKCACPTNFYLGSDGRTCVSNCTASQFVCKNDKCIPFWWKCDTEDDCGDHSDEPPDCPEFKCRPGQFQCSTGICTNPAFICDGDNDCQDNSDEANCDIHVCLPSQFKCTNTNRCIPGIFRCNGQDNCGDGEDERDCPEVTCAPNQFQCSITKRCIPRVWVCDRDNDCVDGSDEPANCTQMTCGVDEFRCKDSGRCIPARWKCDGEDDCGDGSDEPKEECDERTCEPYQFRCKNNRCVPGRWQCDYDNDCGDNSDEESCTPRPCSESEFSCANGRCIAGRWKCDGDHDCADGSDEKDCTPRCDMDQFQCKSGHVIPLRWRCDADADCMDGSDEEACGTGVRTCPLDEFQCNNTLCKPLAWKCDGEDDCGDNSDENPEECARFVCPPNRPFRCKNDRVCLWIGRQCDGTDNCGDGTDEEDCEPPTAHTTHCKDKKEFLCRNQRCLSSSLRCNMFDDCGDGSDEEDCSIDPKLTSCATNASICGDEARCVRTEKAAYCACRSGFHTVPGQPGCQDINECLRFGTCSQLCNNTKGGHLCSCARNFMKTHNTCKAEGSEYQVLYIADDNEIRSLFPGHPHSAYEQAFQGDESVRIDAMDVHVKAGRVYWTNWHTGTISYRSLPPAAPPTTSNRHRRQIDRGVTHLNISGLKMPRGIAIDWVAGNVYWTDSGRDVIEVAQMKGENRKTLISGMIDEPHAIVVDPLRGTMYWSDWGNHPKIETAAMDGTLRETLVQDNIQWPTGLAVDYHNERLYWADAKLSVIGSIRLNGTDPIVAADSKRGLSHPFSIDVFEDYIYGVTYINNRVFKIHKFGHSPLVNLTGGLSHASDVVLYHQHKQPEVTNPCDRKKCEWLCLLSPSGPVCTCPNGKRLDNGTCVPVPSPTPPPDAPRPGTCNLQCFNGGSCFLNARRQPKCRCQPRYTGDKCELDQCWEHCRNGGTCAASPSGMPTCRCPTGFTGPKCTQQVCAGYCANNSTCTVNQGNQPQCRCLPGFLGDRCQYRQCSGYCENFGTCQMAADGSRQCRCTAYFEGSRCEVNKCSRCLEGACVVNKQSGDVTCNCTDGRVAPSCLTCVGHCSNGGSCTMNSKMMPECQCPPHMTGPRCEEHVFSQQQPGHIASILIPLLLLLLLVLVAGVVFWYKRRGQGAKGFQHQRMTNGAMNVEIGNPTYKMYEGGEPDDVGGLLDADFALDPDKPTNFTNPVYATLYMGGHGSRHSLASTDEK RELLGRGPEDEIGDPLALRP1 amino acids 1-172 (SEQ ID NO: 2)IDAPKTCSPKQFACRDQITCISKGWRCDGERDCPDGSDEAPEICPQSKAQRCQPNEHNCLGTELCVPMSRLCNGVQDCMDGSDEGPHCRELQGNCSRLGCQHHCVPTLDGPTCYCNSSFQLQADGKTCKDFDECSVYGTCSQLCTNTDGSFICGCVEGYLLQPDNRSCKAKN LRP1 amino acids 140-164 (SEQ ID NO: 18)YGTCSQLCTNTDGSFICGCVEGYLL LRP1 amino acids 151-172 (SEQ ID NO: 3)FICGCVEGYLLQPDNRSCKAKN LRP1 amino acids 151-164 (SEQ ID NO: 19)FICGCVEGYLL

In some embodiments, the agent that binds to the binding domain I ofLRP1 is a protein or peptide. In some embodiments, the agent that bindsto the binding domain I of LRP1 is an antibody. In some embodiments, theagent that binds to the binding domain I of LRP1 is an antibodyfragment. In some embodiments, the agent that bind to binding domain Iof the LRP protein (e.g., amino acids 1-172) is a small molecule. Oneskilled in the art is familiar with methods of identifying smallmolecules that bind to any protein or peptide.

The agent described herein (e.g., an antibody or a small molecule), whenbinds to binding domain I of the LRP1 protein (e.g., amino acids 1-172),activates a Rho-GTPase pathway. In some embodiments, the Rho-GTPasepathway is mediated by LRP-1. This is based, at least in part, on thefindings of the present disclosure that a prosaposin-derived peptideactivates the Rho-GTPase pathway in a LRP1-dependent manner (e.g., FIG.8A), which in turn stimulates Tsp-1 and suppresses cancer, and that theprosaposin-derived peptide binds to LRP1 in binding domain I (e.g.,FIGS. 13A and 13B).

A “Rho-GTPase” is a molecular switch that controls a wide variety ofsignal transduction pathways in all eukaryotic cells. Rho GTPases playimportant roles in regulating the actin cytoskeleton, regulating cellpolarity, microtubule dynamics, membrane transport pathways, andtranscription. A “Rho-GTPase pathway” refers to a signal transductionpathway that is regulated by a Rho GTPase. “Activate a Rho-GTPasepathway” means the intensity of a signaling pathway regulated by a RhoGTPase is enhanced by at least 30% (e.g., at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, at least 2-fold, at least 3-fold, at least 5-fold, at least10-fold, at least 20-fold. at least 50-fold, at least 100-fold, or more)after the activation, compared to before.

“Stimulate,” as used herein, means to activate or to increase the levelor activity of a biological molecule (e.g., a protein). For example, theagent of the present disclosure “stimulates Tsp-1” means the expressionlevel or activity level of Tsp1 is increased by at least 30% (e.g., atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, at least 2-fold, at least3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least50-fold, at least 100-fold, or more) in the presence of the agent, ascompared to without the agent.

Further provided herein are agents that inhibit function of PRSS2. “Thefunction of PRSS2” refers to its protease function and any otherbiological function it has. Known biological function of PRSS2 include,but are not limited to: (i) upregulation in a subject with pancreatitis;(ii) activating pro-urokinase in ovarian tumors; (iii) cleaving type IIcollagen triple helix in rheumatoid arthritis synovitis tissue; and (iv)degrading type II collagen-rich cartilage matrix. it is described hereinthat PRSS2 binds to LRP1 and represses Tsp-1.

In some embodiments, the agent may inhibit the ability of PRSS2 torepress Tsp-1. In some embodiments, the agent inhibits the expression ofPRSS2. In some embodiments, the agent inhibits the binding of PRSS2 toLRP1 (e.g., to binding domain I of LRP1). In some embodiments, the agentdoes not inhibit the enzymatic activity (protease activity) of PRSS2.

“Inhibit,” as used herein, means to prevent expression, to reduce thelevel of a protein PRSS2), or to decrease the activity of a biologicalmolecule (e.g., a protein). For example, an agent that inhibits theexpression of PRSS2 may prevent PRSS2 from being expressed, or it mayreduce the level of PRSS2 by at least 30% (e.g., by at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or more), compared to in theabsence of the agent. An agent that inhibits the binding of PRSS2 toLRP1 may reduce the amount of PRSS2 that binds to LRP1 by (e.g., by atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 99%, or more), comparedto in the absence of the agent.

Agents that inhibit the expression of a protein is known in the art. Forexample, protein expression may be inhibited by RNA interference (RNAi).“RNA interference (RNAi) is a biological process in which RNA moleculesinhibit gene expression or translation, by neutralizing targeted mRNAmolecules. In some embodiments, the agent is a microRNA, a smallinterfering RNA (siRNA), or a short hairpin RNA (shRNA) that inhibitsthe expression of PRSS2. A “microRNA” is a small non-coding RNA molecule(containing about 22 nucleotides) that functions in RNA silencing andpost-transcriptional regulation of gene expression. A “siRNA” is acommonly used RNA interference (RNAi) tool for inducing short-termsilencing of protein coding genes. siRNA is a synthetic RNA duplexdesigned to specifically target a particular mRNA for degradation. A“shRNA” an artificial RNA molecule with a tight hairpin turn that can beused to silence target gene expression via RNA interference (RNAi).Expression of shRNA in cells is typically accomplished by delivery ofplasmids or through viral or bacterial vectors. In some embodiments, theshRNA that inhibits the expression of PRSS2 comprises the nucleotidesequence of CCGGTCTGAGTTCTGGTGCCGACTACTCGAGTAGTCGGCACCAGAACTCAGATTTT TG(SEQ ID NO: 4). The exemplary shRNA sequence is not meant to belimiting. One skilled in the art is familiar with methods of genesilencing using any of the RNA molecules described herein.

In some embodiments, the agent inhibits binding of PRSS2 to LRP1. insome embodiments, inhibiting binding of PRSS2 to LRP1 inhibits therepression of Tsp-1 by PRSS2. “Inhibits the repression of Tsp-1” meansthat the agent reduces the repression of PRSS2 on Tsp-1 expression oractivity by at least 30% (e.g., by at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 99%, or more), compared to in the absence of the agent.

In some embodiments, an agent that inhibits binding of PRSS2 to LRP1 isa protein or a peptide that binds to PRSS2. In some embodiments, theprotein or peptide is derived from binding domain I of LRP1 (e.g., aminoacids 1-172 of LRP1). For example, the protein or peptide that inhibitsbinding of PRSS2 to LRP1 may comprise an amino acid sequence that is atleast 80% identical to the amino acid sequence corresponding to aminoacids 1-200, 1-199, 1-198, 1-197, 1-196, 1-195, 1-194, 1-193, 1-192,1-191, 1-190, 1-189, 1-188, 1-187, 1-186, 1-185, 1-184, 1-183, 1-182,1-181, 1-180, 1-179, 1-178, 1-177, 1-176, 1-175, 1-174, 1-173, 1-172,1-171, 1-170, 1-201, 1-202, 1-203, 1-204, 1-205, 1-206, 1-207, 1-208,1-209, 1-210, 1-211, 1-212, 1-213, 1-214, 1-215, 1-216, 1-217, 1-218,1-219, 1-220, 1-221, 1-222, 1-223, 1-224, 1-225, 1-226, 1-227, 1-228,1-229, or 1-230 of the LRP1 protein. In some embodiments, the protein orpeptide that inhibits binding of PRSS2 to LRP1 may comprise an aminoacid sequence that is at least 80%, at least 85%, at least 90%, at least95%, at least 99% or more identical to amino acid sequence correspondingto amino acids 1-200, 1-199, 1-198, 1-197, 1-196, 1-195, 1-194, 1-193,1-192, 1-191, 1-190, 1-189, 1-188, 1-187, 1-186, 1-185, 1-184, 1-183,1-182, 1-181, 1-180, 1-179, 1-178, 1-177, 1-176, 1-175, 1-174, 1-173,1-172, 1-171, 1-170, 1-201, 1-202, 1-203, 1-204, 1-205, 1-206, 1-207,1-208, 1-209, 1-210, 1-211, 1-212, 1-213, 1-214, 1-215, 1-216, 1-217,1-218, 1-219, 1-220, 1-221, 1-222, 1-223, 1-224, 1-225, 1-226, 1-227,1-228, 1-229, or 1-230 of the LRP1 protein.

In some embodiments, the protein or peptide that inhibits binding ofPRSS2 to LRP1 comprises an amino acid sequence that is at least 80%(e.g., at least 80%, at least 90%, or 100%) identical to the amino acidsequence corresponding to about amino acids 140-180 of the LRP1 protein.For example, the protein or peptide that inhibits binding of PRSS2 toLRP1 may comprise an amino acid sequence that is at least 80% (e.g., atleast 80%, at least 90%, or 100%) identical to the amino acid sequencecorresponding to amino acids 140-180, 140-179, 140-178, 140-177,140-176, 140-175, 140-174, 140-173, 140-172, 140-171, 140-170, 140-169,140-168, 140-167, 140-166, 140-165, 141-180, 141-179, 141-178, 141-177,141-176, 141-175, 141-174, 141-173, 141-172, 141-171, 141-170, 141-169,141-168, 141-167, 141-166, 142-180, 142-179, 142-178, 142-177, 142-176,142-175, 142-174, 142-173, 142-172, 142-171, 142-170, 142-169, 142-168,142-167, 143-180, 143-179, 143-178, 143-177, 143-176, 143-175, 143-174,143-173, 143-172, 143-171, 143-170, 143-169, 143-168, 144-180, 144-179,144-178, 144-177, 144-176, 144-175, 144-174, 144-173, 144-172, 144-171,144-170, 144-169, 145-180, 145-179, 145-178, 145-177, 145-176, 145-175,145-174, 145-173, 145-172, 145-171, 145-170, 146-180, 146-179, 146-178,146-177, 146-176, 146-175, 146-174, 146-173, 146-172, 146-171, 147-180,147-179, 147-178, 147-177, 147-176, 147-175, 147-174, 147-173, 147-172,148-180, 148-179, 148-178, 148-177, 148-176, 148-175, 148-174, 148-173,149-180, 149-179, 149-178, 149-177, 149-176, 149-175, 149-174, 149-173,149-172, 151-180, 151-179, 151-178, 151-177, 151-176, 151-175, 151-174,151-173, 151-172, 152-180, 152-179, 152-178, 152-177, 152-176, 152-175,152-174, 152-173, 152-172, 153-180, 153-179, 153-178, 153-177, 153-176,153-175, 153-174, 153-173, 153-172, 154-180, 154-179, 154-178, 154-177,154-176, 154-175, 154-174, 154-173, 154-172, 156-180, 156-179, 156-178,156-177, 156-176, 156-175, 156-174, 156-173, 156-172, 156-180, 156-179,156-178, 156-177, 156-176, 156-175, 156-174, 156-173, 156-172, 157-180,157-179, 157-178, 157-177, 157-176, 157-175, 157-174, 157-173, 157-172,158-180, 158-179, 158-178, 158-177, 158-176, 158-175, 158-174, 158-173,158-172, 159-180, 159-179, 159-178, 159-177, 159-176, 159-175, 159-174,159-173, 159-172, 160-180, 160-179, 160-178, 160-177, 160-176, 160-175,160-174, 160-173, or 160-172 of the LRP1 protein. In some embodiments,the protein or peptide that inhibits binding of PRSS2 to LRP1 comprisesan amino acid sequence that is at least 80% (e.g., at least 80%, atleast 90%, or 100%) identical to the amino acid sequence correspondingto amino acids 151-172 of the LRP1 protein (SEQ ID NO: 3). In someembodiments, the protein or peptide that inhibits binding of PRSS2 toLRP1 comprises an amino acid sequence that is at least 80% (e.g., atleast 80%, at least 90%, or 100%) identical to the amino acid sequencecorresponding to amino acids 140-164 of the LRP1 protein (SEQ ID NO:18). In some embodiments, the protein or peptide that inhibits bindingof PRSS2 to LRP1 comprises an amino acid sequence that is at least 80%(e.g., at least 80%, at least 90%, or 100%) identical to the amino acidsequence corresponding to amino acids 151-164 of the LRP1 protein (SEQID NO: 19).

In some embodiments, the protein or peptide that inhibits binding ofPRSS2 to LRP1 is an antibody or an antibody fragment. In someembodiments, the antibody is a monoclonal antibody. In some embodiments,the antibody is a polyclonal antibody. In some embodiments, the antibodybinds to a region of PRSS2 where PRSS2 binds LRP-1. In some embodiments,the agent that inhibits PRSS2 function is a small molecule.

The term “bind” refers to the association of two entities (e.g., twoproteins). Two entities (e.g., two proteins) are considered to bind toeach other when the affinity (KD) between them is <10⁻⁴ M, <10⁻⁵ M,<10⁻⁶ M, <10⁻⁷ M, <10⁻⁸ M, <10⁻⁹ M, <10⁻¹⁰ M, <10⁻¹¹ M, or <10⁻¹² M. Oneskilled in the art is familiar with how to assess the affinity of twoentities (e.g., two proteins).

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably herein, and refer to a polymer of amino acid residueslinked together by peptide (amide) bonds. The terms refer to a protein,peptide, or polypeptide of any size, structure, or function. Typically,a protein, peptide, or polypeptide will be at least three amino acidslong. A protein, peptide, or polypeptide may refer to an individualprotein or a collection of proteins. One or more of the amino acids in aprotein, peptide, or polypeptide may be modified, for example, by theaddition of a chemical entity such as a carbohydrate group, a hydroxylgroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. A protein, peptide, or polypeptide may also be asingle molecule or may be a multi-molecular complex. A protein, peptide,or polypeptide may be just a fragment of a naturally occurring proteinor peptide. A protein, peptide, or polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination thereof.

A peptide that is “derived from” a protein (e.g., a peptide derived frombinding domain I of LRP1) means the peptide is obtained from the proteinand has an amino acid sequence that shares homology with the fragment ofthe protein it corresponds to. The amino acid sequence of the peptidemay be at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 99% or 100% identical to the amino acidsequence of the fragment of the protein it corresponds to. A peptidethat is derived from a protein may also contain chemical modifications,amino acid substitutions, and/or unnatural amino acids.

An “antibody” or “immunoglobulin (Ig)” is a large, Y-shaped proteinproduced mainly by plasma cells that is used by the immune system toneutralize an exogenous substance (e.g., a pathogens such as bacteriaand viruses). Antibodies are classified as IgA, IgD, IgE, IgG, and IgM.“Antibodies” and “antibody fragments” include whole antibodies and anyantigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system. An antibody may be a polyclonal antibody or amonoclonal antibody.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical L chains and two H chains (an IgM antibodyconsists of 5 of the basic heterotetramer unit along with an additionalpolypeptide called J chain, and therefore contain 10 antigen bindingsites, while secreted IgA antibodies can polymerize to form polyvalentassemblages comprising 2-5 of the basic 4-chain units along with Jchain). In the case of IgGs, the 4-chain unit is generally about 150,000daltons. Each L chain is linked to a H chain by one covalent disulfidebond, while the two H chains are linked to each other by one or moredisulfide bonds depending on the H chain isotype. Each H and L chainalso has regularly spaced intrachain disulfide bridges. Each H chain hasat the N-terminus, a variable domain (VH) followed by three constantdomains (CH) for each of the α and γ chains and four CH domains for μand ε isotypes. Each L chain has at the N-terminus, a variable domain(VL) followed by a constant domain (CL) at its other end. The VL isaligned with the VH and the CL is aligned with the first constant domainof the heavy chain (CH1). Particular amino acid residues are believed toform an interface between the light chain and heavy chain variabledomains. The pairing of a VH and VL together forms a singleantigen-binding site. For the structure and properties of the differentclasses of antibodies, (e.g., Basic and Clinical Immunology, 8thedition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.),Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6,incorporated herein by reference).

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyis minor differences in CH sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The V domain mediates antigen binding and define specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see, e.g., Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), incorporated herein byreference). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody dependent cellularcytotoxicity (ADCC).

An “antibody fragment” for use in accordance with the present disclosurecontains the antigen-binding portion of an antibody. The antigen-bindingportion of an antibody refers to one or more fragments of an antibodythat retain the ability to specifically bind to an antigen. It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (e.g., as described in Ward et al., (1989)Nature 341:544-546, incorporated herein by reference), which consists ofa VH domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al, (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883, incorporated herein by reference). Suchsingle chain antibodies are also intended to be encompassed within theterm “antigen-binding portion” of an antibody. These antibody fragmentsare obtained using conventional techniques known to those with skill inthe art, and the fragments are screened for utility in the same manneras are full-length antibodies.

In some embodiments, an antibody fragment may be a Fc fragment, a Fvfragment, or a single-change Fv fragment. The Fc fragment comprises thecarboxy-terminal portions of both H chains held together by disulfides.The effector functions of antibodies are determined by sequences in theFc region, which region is also the part recognized by Fc receptors(FcR) found on certain types of cells.

The Fv fragment is the minimum antibody fragment which contains acomplete antigen-recognition and -binding site. This fragment consistsof a dimer of one heavy- and one light-chain variable region domain intight, non-covalent association. From the folding of these two domainsemanate six hypervariable loops (3 loops each from the H and L chain)that contribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

Single-chain Fv also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding (e.g.,as described in Pluckthun in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994); Borrebaeck 1995, incorporated herein by reference).

Antibodies may be isolated. An isolated antibody is one which has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with diagnostic or therapeutic usesfor the antibody, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In preferred embodiments, theantibody will be purified (1) to greater than 95% by weight of antibodyas determined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornon-reducing conditions using Coomassie blue or, preferably, silverstain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

In some embodiments, the antibody of the present disclosure is amonoclonal antibody. A “monoclonal antibody” is an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).Monoclonal antibodies may also be isolated from phage antibodylibraries, e.g., using the techniques described in Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991), incorporated herein by reference.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567 and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc.), and human constant region sequences.

In some embodiments, the antibody of the present disclosure is apolyclonal antibody. A “polyclonal antibody” a mixture of differentantibody molecules which react with more than one immunogenicdeterminant of an antigen. Polyclonal antibodies may be isolated orpurified from mammalian blood, secretions, or other fluids, or fromeggs. Polyclonal antibodies may also be recombinant. A recombinantpolyclonal antibody is a polyclonal antibody generated by the use ofrecombinant technologies. Recombinantly generated polyclonal antibodiesusually contain a high concentration of different antibody molecules,all or a majority of (e.g., more than 80%, more than 85%, more than 90%,more than 95%, more than 99%, or more) which are displaying a desiredbinding activity towards an antigen composed of more than one epitope.

Methods of producing antibodies (e.g., monoclonal antibodies orpolyclonal antibodies) are known in the art. For example, a polyclonalantibody may be prepared by immunizing an animal, preferably a mammal,with an allergen of choice followed by the isolation ofantibody-producing B-lymphocytes from blood, bone marrow, lymph nodes,or spleen. Alternatively, antibody-producing cells may be isolated froman animal and exposed to an allergen in vitro against which antibodiesare to be raised. The antibody-producing cells may then be cultured toobtain a population of antibody-producing cells, optionally after fusionto an immortalized cell line such as a myeloma. In some embodiments, asa starting material B-lymphocytes may be isolated from the tissue of anallergic patient, in order to generate fully human polyclonalantibodies. Antibodies may be produced in mice, rats, pigs (swine),sheep, bovine material, or other animals transgenic for the humanimmunoglobulin genes, as starting material in order to generate fullyhuman polyclonal antibodies. In some embodiments, mice or other animalstransgenic for the human immunoglobulin genes (e.g. as disclosed in U.S.Pat. No. 5,939,598), the animals may be immunized to stimulate the invivo generation of specific antibodies and antibody producing cellsbefore preparation of the polyclonal antibodies from the animal byextraction of B lymphocytes or purification of polyclonal serum.

Monoclonal antibodies are typically made by cell culture that involvesfusing myeloma cells with mouse spleen cells immunized with the desiredantigen (i.e., hybridoma technology). The mixture of cells is dilutedand clones are grown from single parent cells on microtitre wells. Theantibodies secreted by the different clones are then assayed for theirability to bind to the antigen (with a test such as ELISA or AntigenMicroarray Assay) or immuno-dot blot. The most productive and stableclone is then selected for future use.

In some embodiments, the antibodies described herein are “humanized” foruse in human (e.g., as therapeutics). “Humanized” forms of non-human(e.g., rodent) antibodies are chimeric antibodies that contain minimalsequence derived from the non-human antibody. Humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986) Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “small molecule,” as used herein, refers to a molecule of lowmolecular weight (e.g., <900 daltons) organic or inorganic compound thatmay function in regulating a biological process. Nonlimiting examples ofa small molecule include lipids, monosaccharides, second messengers,other natural products and metabolites, as well as drugs and otherxenobiotics.

A “lipid” refers to a group of naturally occurring molecules thatinclude fats, waxes, sterols, fat-soluble vitamins (such as vitamins A,D, E, and K), monoglycerides, diglycerides, triglycerides,phospholipids, and others. A “monosaccharide” refers to a class ofsugars (e.g., glucose) that cannot be hydrolyzed to give a simplersugar. Non-limiting examples of monosaccharides include glucose(dextrose), fructose (levulose) and galactose. A “second messenger” is amolecule that relay signals received at receptors on the cell surface(e.g., from protein hormones, growth factors, etc.) to target moleculesin the cytosol and/or nucleus. Nonlimiting examples of second messengermolecules include cyclic AMP, cyclic GMP, inositol trisphosphate,diacylglycerol, and calcium. A “metabolite” is an molecule that forms asan intermediate produce of metabolism. Non-limiting examples of ametabolite include ethanol, glutamic acid, aspartic acid, 5′ guanylicacid, Isoascorbic acid, acetic acid, lactic acid, glycerol, and vitaminB2. A “xenobiotic” is a foreign chemical substance found within anorganism that is not normally naturally produced by or expected to bepresent within. Non-limiting examples of xenobiotics include drugs,antibiotics, carcinogens, environmental pollutants, food additives,hydrocarbons, and pesticides.

The agent (e.g., an agent that binds to binding domain I of LRP-1 and/orstimulates Tsp-1 or an agent that inhibits the ability of PRSS2 toinhibit Tsp-1) may be formulated in a pharmaceutical composition. Insome embodiments, the pharmaceutical composition further comprises apharmaceutical acceptable carrier. The term “pharmaceutically-acceptablecarrier” as used herein means one or more compatible solid or liquidfiller, diluents or encapsulating substances which are suitable foradministration into a subject, e.g., a human. A pharmaceuticallyacceptable carrier is “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to the tissueof the patient (e.g., physiologically compatible, sterile, physiologicpH, etc.). The term “carrier” denotes an organic or inorganicingredient, natural or synthetic, with which the active ingredient iscombined to facilitate the application. The components of thepharmaceutical compositions also are capable of being co-mingled withthe molecules of the present disclosure, and with each other, in amanner such that there is no interaction which would substantiallyimpair the desired pharmaceutical efficacy. Some examples of materialswhich can serve as pharmaceutically-acceptable carriers include: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium laurel sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. The term “unit dose” when used in reference to apharmaceutical composition of the present disclosure refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The formulation of the pharmaceutical composition may dependent upon theroute of administration. Injectable preparations suitable for parenteraladministration or intratumoral, peritumoral, intralesional orperilesional administration include, for example, sterile injectableaqueous or oleaginous suspensions and may be formulated according to theknown art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution, suspension or emulsion in a nontoxic parenterallyacceptable diluent or solvent, for example, as a solution in 1,3propanediol or 1,3 butanediol. Among the acceptable vehicles andsolvents that may be employed are water, Ringer's solution, U.S.P. andisotonic sodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. The injectable formulations can besterilized, for example, by filtration through a bacterial-retainingfilter, or by incorporating sterilizing agents in the form of sterilesolid compositions which can be dissolved or dispersed in sterile wateror other sterile injectable medium prior to use.

For topical administration, the pharmaceutical composition can beformulated into ointments, salves, gels, or creams, as is generallyknown in the art. Topical administration can utilize transdermaldelivery systems well known in the art. An example is a dermal patch.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the anti-inflammatory agent. Other compositionsinclude suspensions in aqueous liquids or non-aqueous liquids such as asyrup, elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the anti-inflammatory agent, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109, Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono- di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe anti-inflammatory agent is contained in a form within a matrix suchas those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

In some embodiments, the pharmaceutical compositions used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). Alternatively, preservatives can be used toprevent the growth or action of microorganisms. Various preservativesare well known and include, for example, phenol and ascorbic acid. Thecyclic Psap peptide and/or the pharmaceutical composition ordinarilywill be stored in lyophilized form or as an aqueous solution if it ishighly stable to thermal and oxidative denaturation. The pH of thepreparations typically will be about from 6 to 8, although higher orlower pH values can also be appropriate in certain instances.

Other aspects of the present disclosure provide methods of treatingcancer, using the agents and pharmaceutical compositions describedherein. In some embodiments, the method comprises administering to asubject in need thereof an effective amount of an agent that stimulatesTsp-1. In some embodiments, the method comprises administering to thesubject in need there of an agent that binds to binding domain I ofLRP-1 as described herein (e.g., an antibody that targets amino acids151-172 of LRP1). In some embodiments, the method comprisesadministering to the subject in need there of an agent that inhibits theability of PRSS2 to repress Tsp-1. In some embodiments, the methodcomprising administering to the subject in need thereof an effectiveamount of a first agent that inhibits the PRSS2 (e.g., the ability ofPRSS2 to repress Tsp-1), and an effective amount of a second agent thatbinds to binding domain I of LRP1. When more than one agent isadministered, they may be administered simultaneously or sequentially.One skilled in the art (e.g., a physician) is able to determine the modeof administration.

“Treat” or “treatment” of cancer includes, but is not limited to,preventing, reducing, or halting the development of a cancer, reducingor eliminating the symptoms of cancer, suppressing or inhibiting thegrowth of a cancer, preventing or reducing metastasis and/or invasion ofan existing cancer, promoting or inducing regression of the cancer,inhibiting or suppressing the proliferation of cancerous cells, reducingangiogenesis and/or increasing the amount of apoptotic cancer cells.

An “effective amount” is a dosage of an agent sufficient to provide amedically desirable result, such as treatment of cancer. The effectiveamount will vary with the particular disease or disorder being treated,the age and physical condition of the subject being treated, theseverity of the condition, the duration of the treatment, the nature ofany concurrent therapy, the specific route of administration and thelike factors within the knowledge and expertise of the healthpractitioner. For administration to a subject such as a human, a dosageof from about 0.001, 0.01, 0.1, or 1 mg/kg up to 50, 100, 150, or 500mg/kg or more can typically be employed.

In some embodiments, the effective amount is a dosage of an agent thatcauses no toxicity to the subject. In some embodiments, the effectiveamount is a dosage of an agent that causes reduced toxicity to thesubject. Methods for measuring toxicity are welt known in the art (e.g.,biopsy/histology of the liver, spleen, and/or kidney; alaninetransferase, alkaline phosphatase and bilirubin assays for livertoxicity; and creatinine levels for kidney toxicity).

The agents and pharmaceutical compositions described herein can beformulated for a variety of modes of administration, including systemic,topical or localized administration. A variety of administration routesare available. The particular mode selected will depend upon the type ofcancer being treated and the dosage required for therapeutic efficacy.The methods of the disclosure, generally speaking, may be practicedusing any mode of administration that is medically acceptable, meaningany mode that produces effective levels of the active compounds withoutcausing clinically unacceptable adverse effects. Such modes ofadministration include, but are not limited to, oral, rectal, topical,nasal, intradermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Thepharmaceutical compositions described herein are also suitablyadministered by intratumoral, peritumoral, intralesional, intratracheal,intracerebroventricular, intraperitoneal or perilesional routes, toexert local as well as systemic effects.

Techniques and formulations generally can be found in Remington: TheScience and Practice of Pharmacy, Pharmaceutical Press; 22nd edition andother similar references. When administered, a Psap peptide may beapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. Pharmaceutical compositionsand pharmaceutically-acceptable carriers are also described herein. Suchpreparations may routinely contain salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the disclosure. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

In some embodiments, treatment of cancer with the agents orpharmaceutical compositions described may be combined with anothertherapy, such as a chemotherapy agent, radiation, a cytostatic agent, ananti-VEGF agent, an anti-angiogenesis factor, a p53 reactivation agentand/or surgery.

A subject shall mean a human or vertebrate animal or mammal includingbut not limited to a rodent, e.g., a rat or a mouse, dog, cat, horse,cow, pig, sheep, goat, turkey, chicken, and primate, e.g., monkey. Themethods of the present disclosure are useful for treating a subject inneed thereof. A subject in need thereof can be a subject who has a riskof developing cancer (i.e., via a genetic test) or a subject who hascancer.

Subjects having cancer may be identified using any method known in theart (e.g., blood tests, histology, CT scan, X-ray, MRI, physical exam,cytogenitic analysis, urinalysis, or genetic testing). A subjectsuspected of having cancer might show one or more symptoms of thedisease. Signs and symptoms for cancer are well known to those ofordinary skill in the art. Some exemplary laboratory tests include, butare not limited to, testing for cancer biomarkers such as cancer antigen(CA) 15-3, carcinoembryonic antigen (CEA) and HER-2 for breast cancer,human papillomavirus (HPV) E6 and E7 oncoproteins for cervical cancer,alpha-fetoprotein (AFP), AFP fractions L3, P4/5, and the +II band, andultrasonography for hepatocellular carcinoma (HCC), prostate-specificantigen (PSA) for prostate cancer, and serum CA-125 for ovarian and HCC.

The cancer can be benign or malignant, and it may or may not havemetastasized. Any type of cancer is contemplated herein, including, butnot limited to, leukemias, lymphomas, myelomas, carcinomas, metastaticcarcinomas, sarcomas, adenomas, nervous system cancers and genitourinarycancers. Exemplary cancer types include, but are not limited to, adultand pediatric acute lymphoblastic leukemia, acute myeloid leukemia,adrenocortical carcinoma, AIDS-related cancers, anal cancer, cancer ofthe appendix, astrocytoma basal cell carcinoma, bile duct cancer,bladder cancer, bone cancer, biliary tract cancer, osteosarcoma, fibroushistiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma,malignant glioma, glioblastoma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, hypothalamic glioma,breast cancer, male breast cancer, bronchial adenomas, Burkitt lymphoma,carcinoid tumor, carcinoma of unknown origin, central nervous systemlymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer,childhood cancers, chronic lymphocytic leukemia, chronic myelogenousleukemia, acute lymphocytic and myelogenous leukemia, chronicmyeloproliferative disorders, colorectal cancer, cutaneous T-celllymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewingfamily tumors, extracranial germ cell tumor, extragonadal germ celltumor, extrahepatic bile duct cancer, intraocular melanoma,retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinalstromal tumor, extracranial germ cell tumor, extragonadal germ celltumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma,hairy cell leukemia, head and neck cancer, hepatocellular cancer,Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer,hypothalamic and visual pathway glioma, intraocular melanoma, islet celltumors, Kaposi sarcoma, kidney cancer, renal cell cancer, laryngealcancer, lip and oral cavity cancer, small cell lung cancer, non-smallcell lung cancer, primary central nervous system lymphoma, Waldenstrommacroglobulinema, malignant fibrous histiocytoma, medulloblastoma,melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous neckcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, myelodysplastic syndromes, myeloproliferative disorders,chronic myeloproliferative disorders, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer,ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer,pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorialprimitive neuroectodermal tumors, pituitary cancer, plasma cellneoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer,rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterinesarcoma, Sezary syndrome, non-melanoma skin cancer, small intestinecancer, squamous cell carcinoma, squamous neck cancer, supratentorialprimitive neuroectodermal tumors, testicular cancer, throat cancer,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer,trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma,vaginal cancer, vulvar cancer, choriocarcinoma, hematological neoplasm,adult T-cell leukemia, lymphoma, lymphocytic lymphoma, stromal tumorsand germ cell tumors, or Wilms tumor. In some embodiments, the cancer ismelanoma or ovarian cancer.

EXAMPLES

The progression of cancer to the metastatic stage is a majorcontributing factor to its lethality. In order for a tumor to formlethal metastases it must gain access to the vasculature or lymphaticsystem (intravasation), survive during transit, exit the vascular orlymphatic channels (extravasation), and proliferate at the metastaticsite [1]. In this process, heterotypic signaling between the tumor andits microenvironment can affect tumor growth by regulating theproduction and secretion of factors that mediate tumor growth,angiogenesis, and the immune response. Two proteins, prosaposin andPRSS2, were identified through a functional proteomic screen, designedto identify secreted proteins that modulate Tsp-1 in themicroenvironment [2]. Prosaposin is expressed preferentially by weaklymetastatic tumors and stimulates Tsp-1 in the tumor microenvironment.Conversely, PRSS2 is preferentially expressed by highly metastatic cellsand inhibits Tsp-1 expression in the tumor microenvironment. Tsp-1inhibits tumor growth and progression via multi-modal activity,specifically: (1) It is a broadly acting anti-angiogenic factor, (2) Ithas direct anti-tumor activity against tumors that express CD36, and (3)It promotes macrophage phagocytosis and T-cell activation via binding toCD47 [3-5]. The Tsp-1 stimulating activity of prosaposin and the Tsp-1repressing activity of PRSS2 have both been determined to be mediatedvia binding to LRP1. Provided herein are antibodies that mimicprosaposin's Tsp-1 stimulating activity and block the Tsp-1 repressingactivity of PRSS2.

The current standard of care for cancer patients consists of broadlyacting cytotoxic agents (chemotherapy), radiation, and directedtherapeutics that target specific secreted proteins, cell surfacereceptors, or kinases. Historically, there have been two classes oftherapeutics that target the tumor microenvironment, anti-angiogenicdrugs (which have been limited to anti-VEGF therapies) andimmunomodulatory drugs. One of the major drawbacks to therapies thattarget the microenvironment is that they do not have direct anti-tumoractivity and thus their efficacy as monotherapies has been limited.Conversely, a major drawback of targeted therapies and chemotherapiesthat have direct anti-tumor activity is that patients develop resistanceto the drug, in addition to unintended deleterious side effects. Assuch, a therapeutic strategy involving a combination of targetedtherapies/chemotherapy and anti-angiogenic/immunomodulatory drugs hasbeen used with the goal of first shrinking the tumor, via the activityof the anti-tumor drug and then holding the tumor at bay via theactivity of the therapy targeting the tumor microenvironment. Thedevelopment of antibodies that specifically target the tumormicroenvironment to increase the expression of Tsp-1, a protein withpotent anti-tumor, anti-angiogenic and immunomodulatory activity, wouldhave enormous therapeutic potential.

Prosaposin was first identified as a novel suppressor of tumormetastasis, and such inhibition was documented to be achieved bystimulating p53 and subsequently Tsp-1 in the tumor microenvironment[2]. A 5-amino acid cyclic peptide from prosaposin with potentanti-tumor and anti-metastatic activity has since been identified. InAim 1, the binding affinity and activity of the psaptide will beoptimized. In Aim 2, the optimized psaptide will be developed as atherapeutic agent and tested in orthotopic xenograft, syngeneic andspontaneous genetic models of primary and metastatic cancer. In Aim 2,patient tumor tissue samples for CD36, CD47, Tsp-1 and psap expressionwill also be screened to expand the potential indications for thepsaptide. For Aim 2, collaborations will be with the Drapkin lab at DanaFarber Cancer Institute and Harvard Medical School, as well as theAkslen Lab at the Center for Cancer Biomarkers at the University ofBergen, Norway and the Kimmelman Lab at Brigham and Women's Hospital andHarvard Medical School.

The Psaptide Inhibits Primary Pancreatic Tumor Growth and Metastasis

A 4-amino acid peptide derived from prosaposin that stimulates bonemarrow-derived myeloid cells, normally recruited to stimulate tumorgrowth to express the potent anti-angiogenic and anti-tumorigenicprotein Tsp-1, has been identified.

The prosaposin peptide has been shown to inhibit metastasis bystimulating Tsp-1 in bone marrow-derived cells in the tumormicroenvironment [6]. As such, the hypothesis that psap would haveefficacy in treating metastatic pancreatic cancer, a cancer in which themicroenvironment comprises the majority of the tumor mass, was tested[7]. Accordingly, 1×10⁶ AsPc1 human pancreatic cancer cells expressingfirefly luciferase were injected into the pancreas of SCID mice. Thetumors were allowed to grow for 25 days, at which point the luciferaseintensity was greater than 1×10⁸ for all tumors. Treatment was theninitiated with the psaptide at doses of 20 mg/kg and 40 mg/kg QD. Allmice were sacrificed after 21 days of treatment when the control(vehicle) treated mice became moribund. As demonstrated in FIGS. 1 and2A, the psaptide was able to significantly inhibit primary tumor growth.More significantly, upon examination of the liver and spleen, the twomost common sites of metastasis for this cell line and for pancreaticcancer in general, no metastases whatsoever was observed (FIG. 2B).

Multiple Human Cancer Cells Express CD36

Since Tsp-1 elicits its proapoptotic activity by binding to the cellsurface receptor CD36, it was examined whether human cancer cell linesexpress this receptor. A panel of primary human ovarian cancer cells,derived from patient ascites as well as three breast cancer cell linesrepresenting the major subtypes (ER⁺, HER2⁺, and triple negative), wasscreened. Strikingly, all of the cells were found to express easilydetectable levels of CD36 (FIGS. 3A and 3B). From this observation, itwas predicted that the psaptide would have significant efficacy againstovarian cancer by inducing apoptosis in ovarian cancer cells via Tsp-1binding to CD36, and inhibiting angiogenesis. This hypothesis will betested in Aim 1.

Ovarian Cancer Recruit Bone Marrow Derived Myeloid Cells to Ascites thatare the Target of the Peptide.

It was demonstrated that the activity of prosaposin and the therapeuticpeptide are mediated by Gr1⁺/Cd11b⁺ monocytes [6]. As such, the ascitesfluid from mice treated in FIG. 4 was examined for the presence ofGr1⁺/CD11b⁺ cells by FACS analysis. It was observed that the peritonealfluid of the control-treated mice was comprised of >70% Gr1⁺/Cd11b⁺cells, while the fluid in the peritoneal cavity mouse that had been“cured” by peptide treatment was comprised of ˜30% Gr1⁺/CD11b⁺ cells(FIG. 5). This indicates that the ovarian tumor cells were recruitingthese BM-derived cells.

Prosaposin Stimulation of Tsp-1 is Mediated by LRP1

It has been demonstrated that Low Density Lipoprotein Receptor RelatedProtein (LRP) can mediate the uptake of prosaposin [8]. Thus, in orderto determine how prosaposin was able to stimulate the expression ofTsp-1 and p53 in tumor-associated fibroblasts, prostate fibroblasts weretreated with PC3-conditioned media in the presence and absence ofReceptor Associate Protein (RAP), a competitive inhibitor or LRP1binding. Western blot analysis revealed that in the presence of RAP,PC3-conditioned media no longer stimulated Tsp-1 (FIGS. 6A-6B). It hasalso been demonstrated that ligation of LRP1 releases intracellular Ca²⁺stores [9]. To determine whether prosaposin-mediated stimulation ofTsp-1 utilized this pathway, fibroblasts were treated with CM from PC3cells in the presence and absence of the PKC inhibitor Gö 6983. It wasobserved, via western blot analysis, that inhibition of PKC abolishedthe stimulation of Tsp-1 and p53. Thus, it is believed that Prosaposinfunctions via binding to LRP1.

Additionally, LRP1 expression in lung fibroblasts was knocked down withtwo is independent shRNA sequences and the effects on Tsp-1 expressionwere examined. Knocking clown LRP 1 was found to have no effect on Tsp-1expression in the absence of stimulus (FIG. 7). Consistent with theresults obtained from treating cells with RAP, when the lung fibroblastswere treated with conditioned media from either the weakly metastaticPC3 cell line or the metastatic PC3-LIN4 cell line, no stimulation ofTsp-1 was observed, as had been observed in cells transduced with theempty vector (FIG. 7). Strikingly, silencing LRP1 also abrogated therepression LRP1 by LN4-conditioned media, indicating that this activitywas also mediated by a signaling pathway downstream from LRP1 (FIG. 7).

In order to delineate the pathway leading from LRP 1 to Tsp-1stimulation by prosaposin, two pathways that have been reported to bestimulated by LRP1 under different conditions were chosen to beexamined: Rho and Rac [10]. The peptide was observed to stimulate Tsp-1in an LRP1-dependent manner, as co-treatment with RAP blocked thestimulation of Tsp-1 in a dose-dependent fashion (FIG. 8A). Moreover,when lung fibroblasts were treated with the peptide in the presence ofthe Rho Kinase inhibitor Y27632, the stimulation of Tsp-1 was completelyabrogated (FIG. 8B).

Attention was then turned to the mechanism by which PC3M-LN4 cellsrepress Tsp-1 in the tumor microenvironment. In doing so, LN4conditioned media (CM) was fractionated over a Cu²⁺/heparin sepharosecolumn with increasing concentrations of NaCl. The eluted fractions wereused to treat lung fibroblasts and Tsp-1 induction was analyzed bywestern blot. The fractions eluted between 0.9 and 1.0M NaCl were foundto be able to stimulate Tsp-1 (FIG. 9). By analyzing the protein contentof the eluted fractions by tandem liquid chromatography/massspectrometry (LC/MS), a protein was identified that was present in theactive fractions of LN4 CM, but riot present in the adjacent fractionsor the fractions eluted with the same NaCl concentrations from PC3 CM.The identified protein was the serine protease PRSS2. The LC/MS analysiswas validated by performing a western blot of PRSS2 expression in PC3and LN4 cells, which revealed that PRSS2 was expressed at significantlyhigher levels in LN4 cells (FIG. 10). It was then validated that PRSS2was mediating the repression of Tsp-1 by treating lung fibroblasts withLN4 CM in the presence and absence of the serine protease inhibitor,STI, which completely abrogated the repression of Tsp-1 (FIG. 11).Finally, the signaling pathway emanating from LRP1 that was mediatingthe PRSS2-induced repression of Tsp-1 was sought to be delineated. Itwas speculated that if psap was stimulating Tsp-1 via LRP1-mediatedactivation of the Rho pathway, perhaps PRSS2 induced the repression ofTsp-1 via LRP1-mediated activation of the Rae pathway, since the twopathways are antagonistic. By treating WI38 cells with LN4 CM in thepresence and absence of a Rac1 inhibitor, the repression of Tsp-1 wasobserved to be alleviated and Tsp-1 levels were restored to basal levels(FIG. 12). While STI is able to block the repression of Tsp-1 mediatedby PRSS2 due to its lack of specificity for PRSS2 and its ability toinhibit all members of the trypsin family, it does not represent aviable therapeutic strategy. For the same reason, chemical compoundsdesigned to inhibit PRSS2 would also lack specificity due to theconserved nature of the active sites of serine proteases and the trypsinfamily. Therefore, it is believed that an antibody designed to bind toPRSS2 which blocks its ability to repress Tsp-1 will be a potentanti-cancer therapeutic agent.

Finally, the minimal region of the LRP1 receptor that is required forthe stimulation of Tsp-1 by the prosaposin peptide was sought to bedetermined. For these studies, mutants of LRP1 that contained differentregions of the extracellular domain fused to the transmembrane andintracellular domains were utilized (FIG. 13A) [11]. 293T cells, whichexpress very low levels of Tsp-1 and are not stimulated by the peptideto produce Tsp-1, were then transfected with these constructs (FIG.13B). Cyclic psap peptide was found to be able to stimulate Tsp-1 in293T cells transfected with miniLRP1 a and miniLRP1b, but not in cellstransfected with the other miniLRP1 constructs. Strikingly, bothminiLRP1 a and miniLRP1b are the only mutants that contain theN-terminus and the first two β-propeller domains. Based on theseresults, it is believed that prosaposin and the psap peptide stimulateTsp-1 expression via binding to this region of LRP1. An antibody thatbinds to this region of LRP1 is believed to mimic the activity of thepeptide and perhaps bind with even greater affinity and possess improvedpharmacokinetic (PK) and pharmacodynamic (PD) properties.

LRP1 Peptides that Bind to both PSAP and PRSS2

To map the binding sites on LRP1 that binds to prosaposin (PSAP) and/orPRSS2, peptides derived from binding domain I of LRP1 (SEQ ID NOs: 3 and7-18) were tested for their ability to co-immunoprecipitate with PSAP orPRSS2. The results show that peptide 12, (corresponding to amino acids140-164 of LRP1) and peptide 13 (corresponding to amino acids 151-172 ofLRP1) bind to both PRSS2 and PSAP (FIGS. 16A-16B). Peptide 12 andpeptide 13 overlap in the region corresponding to amino acids 151-164 ofLRP1, indicating that the binding site of PRSS2 and PASP is within thisregion of LRP1.

shRNA sequences: PRSS2 TRCN0000046736 (Sigma ID) Sequence:(SEQ ID NO: 4) CCGGTCTGAGTTCTGGTGCCGACTACTCGAGTAGTCGGCACCAGAACTCAGATTTTTG LRP1 1: TRCN0000257134 (Sigma ID) Sequence: (SEQ ID NO: 5)CCGGACAGCTTCCTGAGGGCTAATTCTCGAGAATTAGCCCTCAGGAAGCT GTTTTTTG2: TRCN0000257100 (Sigma ID) Sequence: (SEQ ID NO: 6)CCGGGATCCGTGTGAACCGCTTTAACTCGAGTTAAAGCGGTTCACACGGA TCTTTTTGLRP1 binding domain I and peptide sequencesPeptide 1 (amino acids 1-24 of LRP1): (SEQ ID NO: 7)IDAPKTCSPKQFACRDQITCISKGW Peptide 2 (amino acids 15-39 of LRP1):(SEQ ID NO: 8) RDQITCISKGWRCDGERDCPDGSDEPeptide 3 (amino acids 24-49 of LRP1): (SEQ ID NO: 9)RCDGERDCPDGSDEAPEICPQSKAQ Peptide 4 (amino acids 40-64 of LRP1):(SEQ ID NO: 10) APEICPQSKAQRCQPNENHCLGTELPeptide 5 (amino acids 50-74 of LRP1): (SEQ ID NO: 11)RCQPNENHCLGTELCVPMSRLCNGV Peptide 6 (amino acids 65-89 of LRP1):(SEQ ID NO: 12) TELCVPMSRLCNGVQDCMDGSDEGPPeptide 7 (amino acids 75-99 of LRP1): (SEQ ID NO: 13)QDCMDGSDEGPHCRELQGNCSRLGC Peptide 8 (amino acids 90-114 of LRP1):(SEQ ID NO: 14) HCRELQGNCSRLGCQHHCVPTLDGPPeptide 9 (amino acids 100-126 of LRP1): (SEQ ID NO: 15)QHHCVPTLDGPTCYCNSSFQLQADGKT Peptide 10 (amino acids 115-139 of LRP1):(SEQ ID NO: 16) TCYCNSSFQLQADGKTCKDFDECSVPeptide 11 (amino acids 125-149 of LRP1): (SEQ ID NO: 17)KTCKDFDECSVYGTCSQLCTNTDGSPeptide 12 (amino acids 140-164 of LRP1, binds toboth PRSS2 and prosaposin): (SEQ ID NO: 18) YGTCSQLCTNTDGSFICGCVEGYLLPeptide 13 (amino acids 151-172 of LRP1, binds toboth PRSS2 and prosaposin): (SEQ ID NO: 3) FICGCVEGYLLQPDNRSCKAKN

Mutations in the Active Site of PRSS2 Do Not Affect the Repression ofTsp-1

Wild-type PRSS2 in pCMVSPORT6.1 was mutated using the QuickChangemutagenesis kit: G191R, S200A, S200T and S200C (nucleotide sequencesprovided below). The wild-type and mutant constructs were transfectedinto 293T cells using FuGene transfection reagent. After 48 hours, theconditioned media containing the mutant proteins was harvested and usedto treat WI-38 fibroblasts overnight. Following treatment, the cellswere harvested, lysed and protein concentration determined by Bio-Radprotein assay. Equivalent levels of protein were added to each well andrun on a polyacrylamide SDS gel. Western blotting with antibodiesagainst Tsp-1 and b-actin was then performed using standard protocols.

It was found that the ability of PRSS2 to repress Tsp-1 was notaffected, indicating that binding site for LRP1 is not in the activesite and that antibodies against this region do not affect the proteaseactivity of the enzyme (FIG. 17).

PRSS2 Protein and nucleotide sequencesPRSS2 Protein (Active site: 194-205 200S, G191R inactivating mutation)(SEQ ID NO: 19)MNLLLILTFVAAAVAAPFDDDDKIVGGYICEENSVPYQVSLNSGYHFCGGSLISEQWVVSAGHCYKSAINSKLSGRGCEYHRIQVRLGEHNIEVLEGNEQFINAAKIIRHPKYNSRTLDNDILLIKLSSPAVINSRVSAISLPTAPPAAGTESLISGWGNTLSSGADYPDELQCLDAPVLSQAECEASYPGKITNNMFCVGFLEGGKDSCQGDSGGPVVSNGELQGIVSWGYGCAQKNRPGVYTKVYVYVDWIKDTIAANSPRSS2 Nucleotide WT (Active site: 194-205 200S, G191R inactivating mutation)(SEQ ID NO: 20) ATGAATCTAC TTCTGATCCT TACCTTTGTT GCAGCTGCTG TTGCTGCCCCCTTTGATGAT GATGACAAGA TCGTTGGGGG CTACATCTGT GAGGAGAATTCTGTCCCCTA CCAGGTGTCC TTGAATTCTG GCTACCACTT CTGCGGTGGCTCCCTCATCA GCGAACAGTG GGTGGTGTCA GCAGGTCACT GCTACAAGTCGGCAATTAAC TCAAAATTAT CAGGAAGAGG GTGTGAATAT CACCGCATCCAGGTGAGACT GGGAGAGCAC AACATCGAAG TCCTGGAGGG GAATGAACAGTTCATCAATG CGGCCAAGAT CATCCGCCAC CCCAAATACA ACAGCCGGACTCTGGACAAT GACATCCTGC TGATCAAGCT CTCCTCACCT GCCGTCATCAATTCCCGCGT GTCCGCCATC TCTCTGCCCA CTGCCCCTCC AGCTGCTGGCACCGAGTCCC TCATCTCCGG CTGGGGCAAC ACTCTGAGTT CTGGTGCCGACTACCCAGAC GAGCTGCAGT GCCTGGATGC TCCTGTGCTG AGCCAGGCTGAGTGTGAAGC CTCCTACCCT GAGAAGATTA CCAACAACAT GTTCTGTGTGGGCTTCCTCG AGGGAGGCAA GGATTCCTGC CAGGGTGATT CTGGTGGCCCTGTGGTCTCC AATGGAGAGC TCCAAGGAAT TGTCTCCTGG GGCTATGGCTGTGCCCAGAA GAACAGGCCT GGAGTCTACA CCAAGGTCTA CAACTATGTGGACTGGATTA AGGACACCAT AGCTGCCAAC AGCTAAPRSS2 G191R Nucleotide (Active site: 194-205 200S, G191R inactivating mutation)(SEQ ID NO: 21) ATGAATCTAC TTCTGATCCT TACCTTTGTT GCAGCTGCTG TTGCTGCCCCCTTTGATGAT GATGACAAGA TCGTTGGGGG CTACATCTGT GAGGAGAATTCTGTCCCCTA CCAGGTGTCC TTGAATTCTG GCTACCACTT CTGCGGTGGCTCCCTCATCA GCGAACAGTG GGTGGTGTCA GCAGGTCACT GCTACAAGTCGGCAATTAAC TCAAAATTAT CAGGAAGAGG GTGTGAATAT CACCGCATCCAGGTGAGACT GGGAGAGCAC AACATCGAAG TCCTGGAGGG GAATGAACAGTTCATCAATG CGGCCAAGAT CATCCGCCAC CCCAAATACA ACAGCCGGACTCTGGACAAT GACATCCTGC TGATCAAGCT CTCCTCACCT GCCGTCATCAATTCCCGCGT GTCCGCCATC TCTCTGCCCA CTGCCCCTCC AGCTGCTGGCACCGAGTCCC TCATCTCCGG CTGGGGCAAC ACTCTGAGTT CTGGTGCCGACTACCCAGAC GAGCTGCAGT GCCTGGATGC TCCTGTGCTG AGCCAGGCTGAGTGTGAAGC CTCCTACCCT GAGAAGATTA CCAACAACAT GTTCTGTGTGGGCTTCCTCG AGCGAGGCAA GGATTCCTGC CAGGGTGATT CTGGTGGCCCTGTGGTCTCC AATGGAGAGC TCCAAGGAAT TGTCTCCTGG GGCTATGGCTGTGCCCAGAA GAACAGGCCT GGAGTCTACA CCAAGGTCTA CAACTATGTGGACTGGATTA AGGACACCAT AGCTGCCAAC AGCTAAPRSS2 S200A (Active site: 194-205 200A) (SEQ ID NO: 22)ATGAATCTAC TTCTGATCCT TACCTTTGTT GCAGCTGCTG TTGCTGCCCCCTTTGATGAT GATGACAAGA TCGTTGGGGG CTACATCTGT GAGGAGAATTCTGTCCCCTA CCAGGTGTCC TTGAATTCTG GCTACCACTT CTGCGGTGGCTCCCTCATCA GCGAACAGTG GGTGGTGTCA GCAGGTCACT GCTACAAGTCGGCAATTAAC TCAAAATTAT CAGGAAGAGG GTGTGAATAT CACCGCATCCAGGTGAGACT GGGAGAGCAC AACATCGAAG TCCTGGAGGG GAATGAACAGTTCATCAATG CGGCCAAGAT CATCCGCCAC CCCAAATACA ACAGCCGGACTCTGGACAAT GACATCCTGC TGATCAAGCT CTCCTCACCT GCCGTCATCAATTCCCGCGT GTCCGCCATC TCTCTGCCCA CTGCCCCTCC AGCTGCTGGCACCGAGTCCC TCATCTCCGG CTGGGGCAAC ACTCTGAGTT CTGGTGCCGACTACCCAGAC GAGCTGCAGT GCCTGGATGC TCCTGTGCTG AGCCAGGCTGAGTGTGAAGC CTCCTACCCT GAGAAGATTA CCAACAACAT GTTCTGTGTGGGCTTCCTCG AGGGAGGCAA GGATTCCTGC CAGGGTGATG CTGGTGGCCCTGTGGTCTCC AATGGAGAGC TCCAAGGAAT TGTCTCCTGG GGCTATGGCTGTGCCCAGAA GAACAGGCCT GGAGTCTACA CCAAGGTCTA CAACTATGTGGACTGGATTA AGGACACCAT AGCTGCCAAC AGCTAAPRSS2 S200T Nucleotide (Active site: 194-205 200T) (SEQ ID NO: 23)ATGAATCTAC TTCTGATCCT TACCTTTGTT GCAGCTGCTG TTGCTGCCCCCTTTGATGAT GATGACAAGA TCGTTGGGGG CTACATCTGT GAGGAGAATTCTGTCCCCTA CCAGGTGTCC TTGAATTCTG GCTACCACTT CTGCGGTGGCTCCCTCATCA GCGAACAGTG GGTGGTGTCA GCAGGTCACT GCTACAAGTCGGCAATTAAC TCAAAATTAT CAGGAAGAGG GTGTGAATAT CACCGCATCCAGGTGAGACT GGGAGAGCAC AACATCGAAG TCCTGGAGGG GAATGAACAGTTCATCAATG CGGCCAAGAT CATCCGCCAC CCCAAATACA ACAGCCGGACTCTGGACAAT GACATCCTGC TGATCAAGCT CTCCTCACCT GCCGTCATCAATTCCCGCGT GTCCGCCATC TCTCTGCCCA CTGCCCCTCC AGCTGCTGGCACCGAGTCCC TCATCTCCGG CTGGGGCAAC ACTCTGAGTT CTGGTGCCGACTACCCAGAC GAGCTGCAGT GCCTGGATGC TCCTGTGCTG AGCCAGGCTGAGTGTGAAGC CTCCTACCCT GAGAAGATTA CCAACAACAT GTTCTGTGTGGGCTTCCTCG AGGGAGGCAA GGATTCCTGC CAGGGTGATA CTGGTGGCCCTGTGGTCTCC AATGGAGAGC TCCAAGGAAT TGTCTCCTGG GGCTATGGCTGTGCCCAGAA GAACAGGCCT GGAGTCTACA CCAAGGTCTA CAACTATGTGGACTGGATTA AGGACACCAT AGCTGCCAAC AGCTAAPRSS2 S200C Nucleotide (Active site: 194-205 200C) (SEQ ID NO: 24)ATGAATCTAC TTCTGATCCT TACCTTTGTT GCAGCTGCTG TTGCTGCCCCCTTTGATGAT GATGACAAGA TCGTTGGGGG CTACATCTGT GAGGAGAATTCTGTCCCCTA CCAGGTGTCC TTGAATTCTG GCTACCACTT CTGCGGTGGCTCCCTCATCA GCGAACAGTG GGTGGTGTCA GCAGGTCACT GCTACAAGTCGGCAATTAAC TCAAAATTAT CAGGAAGAGG GTGTGAATAT CACCGCATCCAGGTGAGACT GGGAGAGCAC AACATCGAAG TCCTGGAGGG GAATGAACAGTTCATCAATG CGGCCAAGAT CATCCGCCAC CCCAAATACA ACAGCCGGACTCTGGACAAT GACATCCTGC TGATCAAGCT CTCCTCACCT GCCGTCATCAATTCCCGCGT GTCCGCCATC TCTCTGCCCA CTGCCCCTCC AGCTGCTGGCACCGAGTCCC TCATCTCCGG CTGGGGCAAC ACTCTGAGTT CTGGTGCCGACTACCCAGAC GAGCTGCAGT GCCTGGATGC TCCTGTGCTG AGCCAGGCTGAGTGTGAAGC CTCCTACCCT GAGAAGATTA CCAACAACAT GTTCTGTGTGGGCTTCCTCG AGGGAGGCAA GGATTCCTGC CAGGGTGATT GTGGTGGCCCTGTGGTCTCC AATGGAGAGC TCCAAGGAAT TGTCTCCTGG GGCTATGGCTGTGCCCAGAA GAACAGGCCT GGAGTCTACA CCAAGGTCTA CAACTATGTGGACTGGATTA AGGACACCAT AGCTGCCAAC AGCTAA

REFERENCES

-   1. Fidler, I. J., The pathogenesis of cancer metastasis: the ‘seed    and soil’ hypothesis revisited. Nat Rev Cancer, 2003. 3(6): p.    453-8.-   2. Kang, S. Y., et al., Prosaposin inhibits tumor metastasis via    paracrine and endocrine stimulation of stromal p53 and Tsp-1. Proc    Natl Acad Sci USA, 2009. 106(29): p. 12115-20.-   3. Lamy, L., et al., Interactions between CD47 and thrombospondin    reduce inflammation. Journal of Immunology (Baltimore, Md.:    1950), 2007. 178(9): p. 5930-9.-   4. Salajegheh, M., et al., Upregulation of thrombospondin-1(TSP-1)    and its binding partners, CD36 and CD47, in sporadic inclusion body    myositis. J Neuroimmunol. 2007. 187(1-2): p. 166-74.-   5. Vallejo, A. N., et al., Central role of thrombospondin-1 in the    activation and clonal expansion of inflammatory T cells. Journal of    Immunology (Baltimore, Md.: 1950), 2000. 164(6): p. 2947-54.-   6. Catena, R., et al., Bone marrow-derived Gr1+ cells can generate a    metastasis-resistant microenvironment via induced secretion of    thrombospondin-1. Cancer Discov, 2013. 3(5): p. 578-89.-   7. Feig, C., et al., The pancreas cancer microenvironment. Clin    Cancer Res, 2012. 18(16): p. 4266-76.-   8. Hiesberger, T., et al., Cellular uptake of saposin (SAP)    precursor and lysosomal delivery by the low density lipoprotein    receptor-related protein (LRP). Embo J, 1998. 17(16): p. 4617-25.-   9. Misra, U. K., G. Gawdi, and S. V. Pizzo, Ligation of low-density    lipoprotein receptor-related protein with antibodies elevates    intracellular calcium and inositol 1,4,5-trisphosphate in    macrophages. Arch Biochem Biophys, 1999. 372(2): p. 238-47.-   10. Mantuano, E., et al., Low density lipoprotein receptor-related    protein (LRP1) regulates Rac1 and RhoA reciprocally to control    Schwann cell adhesion and migration. J Biol Chem, 2010. 285(19): p.    14259-66.-   11. Mikhailenko, I., et al., Recognition of alpha 2-macroglobulin by    the low density lipoprotein receptor-related protein requires the    cooperation of two ligand binding cluster regions. J Biol    Chem, 2001. 276(42): p. 39484-91.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present disclosure toits fullest extent. The specific embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever. All publications cited herein areincorporated by reference for the purposes or subject matter referencedherein.

The indefinite articles “a” and “an” as used herein in the specificationand in the claims, unless clearly indicated to the contrary, should beunderstood to mean “at least one.”

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present disclosure, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the disclosure to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed:
 1. A method of treating cancer, the method comprisingadministering to a subject in need thereof an effective amount of anagent that binds to binding domain I of Low Density LipoproteinReceptor-related Protein 1 (LRP1).
 2. The method of claim 1, whereinbinding domain I of LRP1 comprises amino acids 1-172 of LRP1.
 3. Themethod of claim 1 or claim 2, wherein the agent is a protein or peptide.4. The method of claim 3, wherein the agent is an antibody.
 5. Themethod of claim 4, wherein the antibody is a polyclonal antibody.
 6. Themethod of claim 5, wherein the antibody is a monoclonal antibody.
 7. Themethod of claim 5 or claim 6, wherein the antibody binds to amino acids151-172 (SEQ ID NO: 3) in the binding domain I of LRP1.
 8. The method ofany one of claims 5-7, wherein the antibody binds to amino acids 140-164(SEQ ID NO: 18) in the binding domain I of LRP1.
 9. The method of anyone of claims 5-7, wherein the antibody binds to amino acids 151-164(SEQ ID NO: 19) in the binding domain I of LRP1.
 10. The method of claim1, wherein the agent is a small molecule.
 11. The method of claim 10,wherein the small molecule is selected from the group consisting of:lipids, monosaccharides, second messengers, metabolites, andxenobiotics.
 12. The method of any one of claims 1-11, wherein bindingof the agent to binding domain I of LRP1 activates a Rho-GTPase pathway.13. The method of claim 12, wherein the Rho pathway is a LRP-1 mediatedRho-GTPase pathway.
 14. The method of any one of claims 1-13, whereinbinding of the agent to binding domain I of LRP1 stimulatesThrombospondin 1 (Tsp-1).
 15. The method of any one of claims 1-14,wherein the agent is administered orally, parenterally, intramuscularly,intranasally, intratracheally, intracerebroventricularly, intravenously,or intraperitoneally.
 16. The method of any one of claims 1-15, whereinthe cancer is metastatic.
 17. The method of any one of claims 1-16,wherein the cancer is biliary tract cancer; bladder cancer; braincancer; glioblastoma; medulloblastoma; breast cancer; cervical cancer;choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer;gastric cancer; hematological neoplasm; acute lymphocytic andmyelogenous leukemia; multiple myeloma; AIDS-associated leukemias andadult T-cell leukemia, lymphoma; intraepithelial neoplasm; Bowen'sdisease; Paget's disease; liver cancer; lung cancer; lymphomas;Hodgkin's disease; lymphocytic lymphoma; neuroblastomas; oral cancer;squamous cell carcinoma; ovarian cancer; pancreatic cancer; prostatecancer; rectal cancer; sarcomas; leiomyosarcoma; rhabdomyosarcoma;liposarcoma; fibrosarcoma; osteosarcoma; skin cancer; testicular cancer;stromal tumors and germ cell tumors; thyroid cancer; and renal cancer.18. The method of claim 17, wherein the cancer is prostate cancer,breast cancer, ovarian cancer, or pancreatic cancer.
 19. An antibodythat binds binding domain I of Low Density Lipoprotein Receptor-relatedProtein 1 (LRP1).
 20. The antibody of claim 19, wherein binding domain Iof LRP-1 comprises the amino acids 1-172 of LRP-1.
 21. The antibody ofclaim 19 or claim 20, wherein the antibody binds to amino acids 151-172(SEQ ID NO: 3) in binding domain I of LRP1.
 22. The method of any one ofclaims 19-21, wherein the antibody binds to amino acids 140-164 (SEQ IDNO: 18) in the binding domain I of LRP1.
 23. The method of any one ofclaims 19-21, wherein the antibody binds to amino acids 151-164 (SEQ IDNO: 19) in the binding domain I of LRP1.
 24. The antibody of any one ofclaims 19-23, wherein the antibody is a polyclonal antibody.
 25. Theantibody of any one of claims 19-23, wherein the antibody is amonoclonal is antibody.
 26. The antibody of any one of claims 19-25,wherein the antibody stimulates Tsp-1.
 27. A method of treating cancer,the method comprising administering to a subject in need thereof aneffective amount of an agent that stimulates Thrombospondin 1 (Tsp-1).28. The method of claim 27, wherein the agent inhibits the ability ofProtease, serine 2 (PRSS2) to repress Tsp-1.
 29. The method of claim 27,wherein the agent binds to binding domain I of LRP1.
 30. The method ofany one of claims 26-29, wherein the agent inhibits binding of PRSS2 toLow density lipoprotein receptor-related protein 1 (LRP1).
 31. A methodof treating cancer, the method comprising administering to a subject inneed thereof an effective amount of a first agent that inhibits thefunction of Protease, serine 2 (PRSS2), and an effective amount of asecond agent that binds to binding domain I of Low density lipoproteinreceptor-related protein 1 (LRP1).
 32. The method of claim 31, whereinthe first agent and the second agent are administered simultaneously.33. The method of claim 31, herein the first agent and the second agentare administered sequentially.